GVHD is initiated by small numbers of T cells transferred in the donor marrow that respond to host alloantigens. Donor T cell depletion significantly reduces the incidence and severity of GVHD but increases graft failure, opportunistic infections, EBV induced lymphoproliferative disease, and loss of graft vs tumor effect. Moreover, these complications become more prevalent and severe with increasing donor; host disparity. Many transplant centers have abandoned non-specific T cell depletion and have returned to non- specific immunosuppression to prevent and control GVHD. The central goal of this project is to attempt to selectively inactivate only the small numbers of allospecific T cells transferred in the donor BM that are responsible for GVH. Alloractive T cells require two signals for activation. One signal is delivered by alloAg via the TCR and the other by costimulatory molecules. Blockade of B7 family mediated costimulation can induce anergy to fully mismatched allogeneic donor T cells ex vivo. Based on these findings, we have commenced a clinical trial of ex vivo tolerance induction of donor T cells to alloAg. If long- lasting and irreversible unresponsiveness to alloAg can be induced, the associated attendant clinical toxicities of GVHD would be ameliorated and the eligible donor pool increased without sacrificing immunity to infectious agents and tumor. Although our clinical experiment is ongoing, we believe that success in the clinic will ultimately require greater knowledge about the molecular basis of the state of anergy and the biochemical pathways responsible for its induction and prevention. Moreover, the knowledge gained from this project will likely be important to the success of solid organ transplantation where the confounding variables of hematopoietic alloengraftment and immune reconstitution are not operative. To achieve these goals, three Aims are proposed. First, to determine whether additional molecules can prevent the induction of anergy and to define the phenotype of the anergized T cell. Second, to induce MHC class I specific alloantigen specific anergy in CD8plus T cells. Third, to identify the biochemical and molecular events associated with alloantigen specific T cell anergy. These Specific Aims are designed not only to attempt to extend our fundamental knowledge about the cellular and biochemical events responsible for the anergy but specifically to provide guidance to the first and third projects in their selection of the pathways that must be blocked ex vivo and/or in vivo to achieve our clinical objectives.